PROJECT SUMMARY Current influenza (flu) vaccination inducing strain-specific immunity against variable hemagglutinin (HA) proteins is suboptimal for providing cross protection against antigenically-different epidemic strains and unanticipated new pandemic viruses. Development of an effective flu vaccine inducing enhanced cross protection against viruses with different HA variants is a high priority. M2 protein extracellular domain (M2e) is highly conserved among human flu A viruses and has been demonstrated to be a promising target for developing cross protective flu A vaccines. However, M2e immunity alone would not be sufficient for providing high efficacy of cross protection. Neuraminidase (NA) is the 2nd major glycoprotein on the flu virus, an antiviral drug target, and much less variable than HA. NA immunity was implicated to independently correlate with enhanced protection alleviating disease in humans. Inactivated split flu vaccination was not effective in inducing protective NA immune responses probably due to immunodominant HA intra-vironic suppressive effects. We developed cross protective vaccine candidate containing tandem repeat of M2e on virus-like particles (5xM2e VLP) and VLP was found to be a highly immunogenic platform. M2e repeat 5xM2e VLP was found to be effective in broadening cross protection as standalone, in supplemented (M2e+HA) vaccination, and under a pre-existing flu immunity. We also generated NA VLP and found that NA immunity could be significantly more effective than HA-based inactivated split vaccination in providing cross protection and M2e+NA combined immunity conferred additively enhanced cross protection. In this 1-year STTR phase 1 project, we propose to develop multi subtypes NA plus 5xM2e VLP as an effective cross protective vaccine candidate in a single entity. The proof-of-concept phase 1 project will test a hypothesis that VLP vaccines inducing immunity to M2e and multi subtypes NA as standalone or in supplemented split (HA) vaccination will enhance the efficacy of cross protection against flu viruses. In the specific aim 1, we will determine the immunogenicity and efficacy of cross protection by multi subtypes NA plus 5xM2e presented on the single VLP in mice. In the aim 2 studies, we will determine cross protective efficacy of multi subtype NA plus 5xM2e VLP vaccine and supplemented flu vaccination in ferrets. Premise and rigor are strengthened by proposing flu vaccine efficacy studies using both preclinical mouse and ferret models through collaboration with experienced investigators. The outcomes and data from the phase 1 project are expected to provide sufficient data supporting the advanced STTR phase 2 studies in a future direction, eventually contributing to developing an effective universal flu vaccine.